| Follicular atresia is a normal physiological phenomenon in the follicular development of female mammals and is an important physiological mechanism for selection of healthy follicles.The classfication of the status of follicular atresia and the study of physiological mechanism will hopefully change the fate of atretic follicles and improve the reproduction efficiency of female animals.Therefore,this study explored suitable methods to classify the status of follicular atresia in buffalo.And non-targeted metabolomics was also used to analyze the metabolic characteristics of follicular fluid with different status of atresia,and to screen for differential metabolites with different status of atresia and targeted metabolic pathways related to follicular atresia.These results will lay a foundation for further identification of metabolic biomarkers in the follicular fluid of buffalo atretic follicles,and for understanding of the mechanism of follicular atresia.Study on the the suitable classfication methods of the status of follicular atresia in buffalo.Buffalo follicles were divided into healthy follicles(HF),early atretic follicles(EF)and late atretic follicles(LF)by using three methods:observation of follicle appearance,morphological observation of follicular internal circumstance,estrogen(E2)/progesterone(P4)ratio,respectively.Based on the analysis of the relationship between the single method and the comprehensive method,a classfication method suitable for the status of buffalo follicular atresia was formed.The apoptosis rate of follicular granulosa cells with different status of atresia and the expression level of regulating follicular atresia were detected to further verify the accuracy of the comprehensive classfication method.The results showed:(1)There were obvious internal and external morphological changes in the process of buffalo follicular atresia,morphological observation could be used to classify the status of buffalo follicular atresia.(2)The process of buffalo follicular atresia was accompanied by obvious differences of E2 and P4 concentrations in follicular fluid,and the E2 / P4 ratio could be used to classify the status of atresia more accurately.The judgment standard was HF: E2 / P4> 5;EF: 1 ≦ E2 / P4 ≦ 5;LF: 0<E2 / P4 <1.(3)During the process of buffalo follicle atresia,the changes of each component of the follicle were different.The E2 / P4 ratio combined with the morphological observation of the internal and external follicles to make a comprehensive classfication method,which was more accurate than the single method to classify the status of buffalo follicular atresia.(4)In the comprehensive classfication method,with the extent of atresia deepened,the apoptosis rate of follicular granulosa cells increased significantly(HF:11.35±0.69%,EF:18.53±0.43%,LF:28.78±0.64%,P<0.05).The expression levels of genes FSHR,LHR,CYP19 and Bcl-2 that inhibited follicular atresia were significantly reduced(P<0.05).The expression levels of genes Caspase-3,Caspase-9 and Bax that promoted follicular atresia were significantly increased(P <0.05).Non-targeted metabolomics analysis of buffalo follicular fluid with different status of atresia.The test was based on the liquid chromatography-mass spectrometry(LC-MS)metabolite full-spectrum analysis platform,combined with multidimensional statistical analysis(Principal Component Analysis PCA,Partial Least Squares Discrimination Analysis PLS-DA)and single-dimensional statistical analysis(Student’s t test and Fold Change analysis),the metabolites that had significant difference between groups were screened out.The KEGG pathway enrichment analysis was performed on the differential metabolites of HF vs EF,HF vs LF,and EF vs LF groups to screen the potential targeted pathways for buffalo follicular atresia.The results show that this test successfully identified 60 differential metabolites,such as d-mannose,sphingomyelin,linoleic acid,l-carnitine and dehydroascorbic acid.Among them,27 differential metabolites were detected in the HF and EF groups.Compared with HF,11 metabolites in EF were up-regulated and 16 metabolites were down-regulated;47 differential metabolites were detected in the HF and LF groups.Compared with HF,26 metabolites in LF were up-regulated and 21 metabolites were down-regulated;10 differential metabolites were detected in the EF and LF groups.Compared with EF,3 metabolites in LF were up-regulated and 7 metabolites were down-regulated.KEGG pathway enrichment analysis of differential metabolites found that follicular atresia led to metabolic changes such as follicular fatty acid metabolism disorder,lysosomal membrane permeability,reduced glutathione synthesis and weakened TCA cycle.In conclusion,the E2 / P4 ratio combined with the morphological observation method can eliminate the influence of subjective factors to classify the status of follicular atresia in buffalo accurately;We determined the metabolic characteristics of follicular fluid in buffalo with different status of atresia,and screened out 60 differential metabolites such as d-mannose,sphingomyelin,linoleic acid,l-carnitine and dehydroascorbic acid,sought out the metabolic pathway changes in buffalo follicular atresia,such as follicular fatty acid metabolism disorder,lysosomal membrane permeability,reduced glutathione synthesis and weakened TCA cycle. |
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